Inhibition of smooth muscle cell migration by (R)-amlodipine

ABSTRACT

The R(+) isomer of amlodipine is a potent inhibitor of smooth muscle cell migration despite its lack of calcium channel-blocking activity. It is useful for treating atherosclerosis, re-stenosis after angioplasty and endometriosis.

This is a National Stage filing under 35 USC §371 based on InternationalApplication PCT/EP94/02697 which was filed internationally on Aug. 10,1994.

This invention relates to treatment of medical conditions involvingsmooth muscle cell migration using the R(+) isomer of3-ethyl-5-methyl-2-(2-aminoethoxy-methyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methylpyridine-3,5-dicarboxylate,a compound having the approved non-proprietary name "amlodipine".

Amlodipine is a known calcium channel-blocking agent having vasodilatoryactivity and is currently used, generally in the form of apharmaceutically acceptable salt such as its maleate or besylate, intreatment of hypertension and angina. The compound and its preparationare described in European patent 0089167 B1. Amlodipine is a racemiccompound due to its symmetry at position 4 of the dihydropyridine ringand the R- and S-enantiomers may be prepared by methods described in J.Med. Chem. 1986 29 1696 (Arrowsmith et al.) and European PatentApplication 0331315 A. It was formerly believed that the two resolvedenantiomers consisted of the R-(-) and S-(+) isomers but it hassubsequently been found that these are in fact the S(-) and the R(+)isomer, respectively (see J. Med. Chem., 35, 3341-3344 (1992), Goldmannet al.). It is known that the calcium channel blocking activity ofamlodipine is substantially confined to the S(-) form and the racemicmixture of R(+) and S(-) forms; the R(+) isomer has little or no calciumchannel blocking activity and so is not likely to have significantcardiovascular effects when administered to a patient.

It is known that calcium channel blockers in general tend to inhibitsmooth muscle cell migration. Thus they have been found to impede lesiondevelopment in various animal models of atherosclerosis (seeArteriosclerosis 5; 250 (1985), Willis et al., Arteriosclerosis 6; 237(1986), Sugano et al.); also smooth muscle cell proliferative lesionsfollowing endothelial cell damage by balloon angioplasty are reduced byIsradipine, a calcium channel blocker (see Am. J. Pathol 124, 88-93(1986) Handley et al.). During restenosis following balloon angioplastyand atherogenesis, vascular smooth muscle cells migrate from the mediato the intima where they proliferate. It is believed that the efficacyof calcium channel blockers in animal models of re-stenosis post-balloonangioplasty and atherosclerosis is due to inhibition of vascular smoothmuscle cell migration and subsequent reduction in smooth muscle cellproliferation and neointimal formation.

Thus, calcium channel blockers would be expected to be useful in thetreatment of conditions of smooth muscle cell migration, includingatherosclerosis, re-stenosis after angioplasty and endometriosis.

It has now been discovered, surprisingly and contrary to all existingtheory, that the R(+) isomer of amlodipine, despite its lack of calciumchannel-blocking activity, is a potent inhibitor of smooth muscle cellmigration and its potency in this respect is greater that of the S(-)isomer of amlodipine and some other known calcium channel-blockers. TheR(+) isomer thus provides a means of treating conditions involvingsmooth muscle cell migration without any concomitant cardiovasculareffects.

It is therefore applicable to patients for whom reduction of bloodpressure would be undesirable.

Thus, one aspect of the invention comprises the R(+) isomer ofamlodipine or a pharmaceutically acceptable salt thereof for use in thetreatment of conditions requiring inhibition of vascular smooth musclecell migration.

The invention also provides use of the R(+) isomer of amlodipine or apharmaceutically acceptable salt thereof for making a medicament fortreatment of conditions requiring inhibition of smooth muscle cellmigration.

A further aspect of the invention provides a pharmaceutical compositioncomprising the R(+) isomer of amlodipine or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier ordiluent, said composition being substantially free of calciumchannel-blocking activity.

The invention also provides a method of treating conditions requiringinhibition of smooth muscle cell migration which comprises administeringto the patient an effective amount of the R(+) isomer of amlodipine or apharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts of amlodipine include the maleateand the besylate. The conditions to be treated include atherosclerosis,incipient re-stenosis following angioplasty, and endometriosis. The R(+)isomer of amlodipine may be used in the absence of the S(-) isomer andof any other compound acting as a calcium channel-blocker.

The effect of the R(+) isomer of amlodipine on smooth muscle cellmigration was demonstrated using an aortic explant assay method in whichthe modulation, migration and proliferation of smooth muscle cells areassessed using primary cultures of rabbit aortic smooth muscle cells asdescribed in Atherosclerosis 86 227-237 (1191). In this method uniformpieces of intimal/medial tissue from rabbit aorta were cultured inindividual wells of a well plate. Migration was induced by addition ofplatelet-derived growth factor to the culture. Following a lag phase ofseveral days the smooth muscle cells migrated from the explanted tissueand proliferated.

The distance covered by the outgrowing smooth muscle cells was measured.

This assay was carried out with varying concentrations of test compoundadded to the culture. The compounds thus tested were the maleate saltsof the racemic mixture of R(+) and S(-) amlodipine, the maleate salts ofR(+) and S(-) amlodipine separately and the known calciumchannel-blocking agents nitrendipine and verapamil.

The results obtained are shown in Table 1, in which the percentageinhibition of smooth muscle cell migration for concentrations of testcompound of 1 nanomole and 0.1 nanomole are recorded.

The racemic, R(+) and S(-) forms of amlodipine maleate were also testedfor inhibition of K⁺ -induced rat aortic contraction by the methoddescribed by Burges et al, J. Cardiovasc Pharmacol 9(1) 110-9; The 1C₅₀(50% inhibitory concentration) values in nanomoles are also recorded inTable 1 and afford a measure of calcium channel-blocking activity.

                  TABLE 1                                                         ______________________________________                                                  % inhibition of                                                               SMC migration                                                                            inhibition of K.sup.+  induced                                     from explants                                                                            rat aortic contraction                                   Compound    1 nM    0.1 nM   1C.sup.50 (nM)                                   ______________________________________                                        Amlodipine (racemate)                                                                     33      39       2                                                Amlodipine R (+)                                                                          39      36       1000                                             Amlodipine S (-)                                                                          30      21       1                                                Nitrendipine                                                                              28      14                                                        Verapamil   22      13                                                        ______________________________________                                    

It is evident from these results that the R(+) enantiomer of amlopidineis effective in inhibiting smooth muscle cell migration even though itsactivity as a calcium channel blocker is negligible.

For administration to man in the curative or prophylactic treatment ofconditions involving smooth muscle migration, oral doses of R(+)amlodipine or its salts may be in the range of 2-10 mg daily for anaverage adult patient (weighing 70 kg), that is a range similar to thatused for amlodipine in the treatment of hypertension. However, theabsence of cardiovascular effects allows administration of much largerdoses than would be recommended for the calcium channel-blocking S(-)isomer or the racemate, with a correspondingly greater effect on cellmigration. The oral dose of R(+) amlodipine or a salt thereof for theaverage adult patient may thus be 20 mg or more and up to 100 mg/day, oreven greater. The actual dose used will be determined by a physicianconsidering the age, weight, condition and medical history of thepatient. For a typical adult patient individual tablets or capsules arelikely to contain 1 to 100 mg of active compound, in a suitablepharmaceutical vehicle or carrier. Dosages for intravenousadministration would be in the range of 1-20 mg of active compound persingle dose as required. Thus, according to another aspect of theinvention, there is provided a unit dose of a pharmaceutical compositionsubstantially free of calcium channel-blocking activity containing (fororal administration) from 1 mg to 100 mg, preferably 20 to 100 mg, ofthe R(+) isomer of amlodipine or a pharmaceutically acceptable saltthereof. A further aspect of the invention provides such a unit dose forintravenous administration containing from 1 to 20 mg of the R(+) isomerof amlodipine or salt thereof.

We claim:
 1. A method of treating conditions requiring inhibition ofsmooth muscle cell migration which comprises administering to thepatient an effective amount of the R(+) isomer of amlodipine or apharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the R(+) isomer of amlodipine or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or diluent, said composition being substantially freeof calcium channel-blocking activity.
 3. A unit dose of a compositionaccording to claim 2, for oral administration, containing from 1 mg to100 mg of the R(+) isomer of amlodipine or a pharmaceutically acceptablesalt thereof.
 4. A unit dose according to claim 3, containing at least20 mg of the R(+) isomer of amlodipine or a pharmaceutically acceptablesalt thereof.
 5. A unit dose of a composition according to claim 2 forintravenous administration, containing from 1 mg to 20 mg of the R(+)isomer of amlodipine or a pharmaceutically salt thereof.